(a) Field of the Invention
The present invention relates to synthetic natriuretic peptides possessing diuretic and natriuretic activities of a very high color order as well as vasorelaxant and smooth muscle relaxant properties together with hypotensive activities in normotensive mammals and anti-hypertensive activities in renovascular hypertensive mammals. Those synthetic natriuretic peptides contain 23 amino acid residues, with residues 2-23 having the natural or L-configuration while the amino-terminal residue is derived from an unnatural amino acid having the D-configuration. This invention also relates to a process for preparing the above compounds having either a free carboxylic acid or a carboxylic acid amide group at the carboxy-terminal, as well as to pharmaceutical preparations containing the above synthetic natriuretic peptides in their free state or as salts with pharmaceutically acceptable acids, and to the use of said pharmaceutical preparations as diuretic and/or natriuretic agents, as vasorelaxants and/or smooth muscle relaxants, and as anti-hypertensive agents in mammals suffering from renovascular hypertension.
(b) Description of Prior Art
Numerous papers have been published since A. J. de Bold et al., 1981 Life Sci. 28, 89 reported that extracts of rat myocardial tissue caused a strong and rapid rise in uninary output and an equally dramatic rise in the excretion of sodium and of chloride when administered intraveneously to non-diuretic rats, with a concomitant decrease in blood pressure; the authors concluded that the extracts contained and extremely powerful inhibitor of renal tubular NaCl reabsorption. Evidence for the polypeptide nature of said inhibitor and the results of preliminary purification showing multimodal distribution were presented by de Bold, 1981 Fed. Proc. 40, 554 and 1982 Proc. Soc. Exp. Biol. Med. 170, 133. Trippodo et al., ibid. p. 502 and 1983 Hypertension 5, Suppl. I, I-81 reported that the above inhibitor or atrial natriuretic factor (ANF) was present in rat, rabbit, dog, baboon, and human atrial extracts and found activity mainly in two fractionation ranges, viz., 36000-44000 and 3600-5500 daltons. Currie et al., 1983 Science 221, 71 showed that rat, pig, and human atrial extracts possessed natriuretic and powerful vasorelaxant and smooth muscle relaxant activities. Further studies on the purification and the composition of rat ANF by de Bold et al., 1983 Life Sci. 33, 292, by Grammar et al., 1983 Biochem. Biophys. Res. Comm. 116, 696, and by Thibault et al., 1983 FEBS Letters 164, 286 resulted in the isolation of peptides composed of 49, 36, and 26, 31, and 33 amino acids, respectively. Flynn et al., 1983 Biochem. Biophys. Res. Comm. 117, 859 succeeded in establishing the sequence of a rat ANF composed of 28 amino acid residues; Currie et al., 1984 Science 223, 67, and Geller et al., 1984 Biochem. Biophys. Res. Comm. 120, 333 isolated and determined the sequences of a number of rat atrial peptides ("atriopeptins") containing 19-24 amino acid, see also U.S. Pat. No. 4,964,544 issued Jan. 29, 1985. Misono et al., 1984 Biochem. Biophys. Res. Comm. 119, 524 isolated a rat ANF containing 25 amino acids and determined its sequence; they found that the presence of the disulfide linkage between the two half-cystine residues was essential for natriuretic, diuretic, and vasorelaxant activitives. Seidah et al., 1984 Proc. Natl. Acad. Sci. USA 81, 2640 prepared and sequenced four ANF obtained from rat tissue and determined their sequences; they were found to contain 33, 32, 31, and 26 amino acid residues, respectively; the authors also succeeded in synthesizing said last-named ANF by coupling four fragments thereof by means of classical method, with each of the above fragments having been prepared by solid-phase synthesis. Garcia et al., 1984 Biochem. Biophys . Res. Comm. 119, 685 reported that the native ANF with 26 , amino acids and the above synthetic ANF, both as the C-terminal carboxylic acid and as the corresponding acid amide, had identical vasorelaxant activities; furthermore, Napier et al., 1984 Biochem. Boiphys. Res. Comm. 120, 981 confirmed the results of Seidah et al. cited above with respect to the native ANF having 33, 32, and 31 amino acid residues, respectively, and established that those compounds had free carboxylic acid groups at the C-terminus. Atlas et al., 1984 Nature 309, 717 isolated and sequenced a rat ANF having 24 amino acid residues ("auriculin A") which was also prepared by solid-phase synthesis. A C-terminal extended form thereof having an additional tyrosine residue ("auriculin B") was also detected. Sugiyama et al., 1984 Biochem. Biophys. Res. Comm. 123, 338 prepared by solid-phase synthesis the rat ANF containing 25 amino acids first described by Misono et al. cited above, as well as atriopeptin I having 21 amino acids first described by Currie et al. cited above. Thibault et al., 1984 Biochem. Biophys. Res. Comm. 125, 938 subjected to synthetic ANF having 26 amino acid residues described by Seidah et al. cited above to Edman degradation and obtained the corresponding N-terminal truncated ANF having 25, 24, 23, and 22 amino acids, respectively; digestion of the above synthetic ANF with various carboxypeptidases gave the corresponding C-terminal truncated ANF containing 25, 24, 23, and 21 amino acids, respectively. Kangawa et al., 1984 Biochem. Biophys. Res. Comm. 118, 131 used human atrial tissue as the starting mayterial and succeeded in isolating, sequencing, and synthesizing by a solid-phase procedure a peptide (.alpha.-hANP) containing 28 amino acid residues; .alpha.-hANP was found to have the same sequence as the 28-amino acid peptide obtained from rat atria by Flynn et al. cited above, except that is contained a methionine residue in position 7 (position 110, see below) where the peptide of Flynn et al. and all other ANF obtained from rat atria disclosed in the above references have an isoleucine residue.
Concerning the numbering of individual residues in the various atrial natriuretic peptides (ANP) described in the above references it was customary to number them consecutively starting with the N-terminal residue as No. 1. However, following the successful cloning of rat and of human cDNA and of the respective genes for ANP, it is now the international consensus to number the respective propeptides consecutively from Asn.sup.1 to Tyr.sup.126 so as to establish an unambiguous numbering system.
The synthetic natriuretic peptides (SNP) of this invention are characterized by containing only 23 amino acid residues, of which residues 2-23 have the natural or L-configuration while the amino-terminal residue is that of an unnatural D-amino acid. We have found, surprisingly, that the synthetic natriuretic peptides of this invention possess the full range of biological activities found in known natriuretic peptides having larger numbers of amino acid residues in their respective molecules, and that they are particularly distinguished by possessing natriuretic and aortic vasorelaxant properties of a high order. However, a totally unexpected property of the SNP of this invention is their significant dissociation between diuretic and natriuretic activity on the one hand which is fully present therein, and their vasorelaxant activity on the other hand which is found to be present to a smaller degree. This surprising dissociation of activities exhibited by the above SNP provides important therapeutic advantages, in that the undesirable effects upon heart rate which are a common feature of known atrial natriuretic factors (ANF) or atrial natriuetic peptides (ANP), viz., either bradycardia or tachycardia, are significantly diminished. The fact that the SNP of this invention are composed of only 23 amino acid residues results in the further advantage that their preparation by classical methods, or preferably by solid phase synthesis, in simpler, more easily performed, and more economical than that of known natriuretic peptides. The particular process of this invention offers the additional advantage that the products are obtained in good yields with a high degree of purity, and that they may be produced at will either in the form of their respective free carboxylic acid or as the corresponding carboxylic acid amide. Both types of the synthetic natriuretic peptides of this invention are useful as research tools, and are particularly useful in the practice of medicine for the treatment of patients suffering from pathological conditions associated with abnormalities in their water and electrolyte balances, for example as diuretic and natriuretic agents, as vasorelaxant and/or smooth muscle relaxant agents, or as hypotensive or anti-hypertensive agents, the latter especially in patients suffering from hypertension of renovascular origin, as well as in cases of congestive heart failure and of edema. Moreover, the synthetic natriuretic peptides of this invention offer the added and surprising advantage of showing a longer duration of action than known natriuretic peptides. The fact that many of the native ANF have been obtained as amino-terminal truncated versions of longer peptides seems to indicate that the mammalian organism contains certain proteases which attack the latter peptides from the amino terminus. The longer duration of action shown by the synthetic natriuretic peptides of this invention is attributed to the protection offered against such proteolytic attack by the presence of an unnatural D-amino acid at their respective amino terminus.
In the following text the symbols for the amino acids are according to the IUPAC-IUB recommendations, see Arch. Biochem. Biophys. 115, 1 (1966). The symbols for the protective groups used in he synthesis process are those described in Shrooder and Lubke, "The Peptides", Academic Press, New York and London, 1965, as well as in the current literature. Other abbreviations used are e.g., SNP: synthetic natriuretic peptide(s); ANP: atrial natriuretic peptide(s); ANF: atrial natriuretic factor(s); TFA: trifluoroacetic acid; DMF: dimethylformamide; TEA: triethylamine; DIEA: diisopropylethylamine; HPLC: high pressure liquid chromatography; RP-HPLC: reverse phase high pressure liquid chromatography. Other symbols and/or abbreviations are those commonly used in peptide chemistry and will readily be understood by those skilled in the art. The following terms are Registered Trade Marks or Trade Names: Sephadex; Vydac.